Anti-human Igβ antibody

ABSTRACT

[Means for Solution] An anti-human Igβ antibody comprising a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence of amino acid numbers 50 to 65 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence of amino acid numbers 98 to 108 of SEQ ID NO: 2, a light chain variable region comprising CDR1 consisting of the amino acid sequence of amino acid numbers 24 to 38 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of amino acid numbers 54 to 60 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence of amino acid numbers 93 to 101 of SEQ ID NO: 4, and a heavy chain constant region which is a human Igγ1 constant region having amino acid mutations of S239D, H268D, and L328W.

TECHNICAL FIELD

The present invention relates to a novel anti-human Igβ antibody whichis useful as an active ingredient of a pharmaceutical composition.

BACKGROUND ART

A B cell receptor (BCR) is composed of membrane immunoglobulin (mIg)molecules assembled with heterodimers of Igα (CD79A) and Igβ (CD79B). Anantigen is bound to the mIg and allow the receptors to aggregate, and anIgα/Igβ subunit transmits a signal to the inside of a B cell (Mol.Immunol., Vol. 41, p. 599-613, 2004).

As for a protein family of an Fcγ receptor (FcγR) which is an Fcreceptor against an IgG antibody, FcγRIa (CD64A), FcγRIIa (CD32A), andFcγRIIIa (CD16A) which have immunoactive functions, and FcγRIIb (CD32B)which has immunosuppressive functions have been reported. It has beenreported that when BCR and FcγRIIb on B cells are crosslinked through anIgG immune complex, an activity of the B cells is suppressed and thus aproliferation of the B cells and antibody production are suppressed(Nat. Rev. Immunol., Vol. 10, p. 328-343, 2010; Nat. Rev. Immunol., Vol.8, p. 34-47, 2008; Nat. Rev. Immunol., Vol. 2, p. 580-592, 2002).

It has been reported that control of the activity of B cells throughsuch FcγRIIb is deeply involved in the pathology of autoimmune diseasessuch as rheumatoid arthritis and systemic lupus erythematosus.

As for the relation to rheumatoid arthritis, it has been reported thatin an FcγRIIb knockout mouse, humoral immunity is not appropriatelycontrolled (Nature, Vol. 379, p. 346-349, 1996; J. Immunol., Vol. 163,p. 618-622, 1999) and susceptibility to collagen-induced arthritis isincreased (J. Exp. Med., Vol. 189, p. 187-194, 1999). Further, it hasbeen confirmed that expression of FcγRIIb in memory B cells ofrheumatoid arthritis patients is decreased (J. Immunol., Vol. 190, p.6015-6022, 2013).

As for the relation to systemic lupus erythematosus, it has beenreported that onset of a systemic lupus erythematosus disease issignificantly suppressed in a transgenic mouse in which expression ofFcγRIIb is enhanced specifically in B cells (J. Exp. Med., Vol. 205, p.883-895, 2008). It has been confirmed that in regard to a knockout mouseof FcγRIIb, self-reactive B cells or plasma cells appear and the diseasecondition of systemic lupus erythematosus develops spontaneously(Immunity, Vol. 13, p. 277-285, 2000; J. Exp. Med., Vol. 207, p.2767-2778, 2010). Further, a decrease in expression of FcγRIIb in memoryB cells of systemic lupus erythematosus patients (J. Exp. Med., Vol.203, p. 2157-2164, 2006; J. Immunol., Vol. 178, p. 3272-3280, 2007) andrelevance between genetic polymorphism in a cell transmembrane region ofFcγRIIb and frequency of onsets of systemic lupus erythematosus(Arthritis Rheum., Vol. 46, p. 1242-1254, 2002) have been reported.

Further, suppression of antibody production by controlling an activityof B cells through FcγRIIb is effective for treating an autoimmunedisease in which an autoantibody is related to the pathologicalcondition.

Idiopathic thrombocytopenic purpura is an autoimmune disease in which anautoantibody against platelets of a patient causes platelet destruction(Autoimmun. Rev., Vol. 13, p. 577-583, 2014). It has been reported thatin an animal to which an antiplatelet antibody is administered,thrombopenia is induced (Br. J. Haematol., Vol. 167, p. 110-120, 2014)and a decrease in an autoantibody are effective for the treatment ofidiopathic thrombocytopenic purpura (Ther. Apher. Dial. Vol. 16, p.311-320, 2012; Lupus, Vol. 22, p. 664-674, 2013).

Therefore, if a monoclonal antibody that crosslinks BCR and FcγRIIb andincreases an immunosuppressive function of FcγRIIb can be developed, itis expected that such monoclonal antibody is useful for prevention ortreatment of autoimmune diseases such as rheumatoid arthritis, systemiclupus erythematosus, and idiopathic thrombocytopenic purpura.

As an antibody that crosslinks BCR and FcγRIIb, DART which is abispecific antibody against Igβ and FcγRIIb (Patent Document 1 andNon-Patent Document 1), and anti-CD19 S267E/L328F which has a variableregion binding to CD19 which is a part of a BCR complex and an Fc regionwhose affinity for FcγRIIb is increased (Patent Document 2 andNon-Patent Documents 2 and 3) are reported. Among these, anti-CD19S267E/L328F is specifically examined, and its inhibitory action withrespect to the activity of B cells in which BCR is stimulated and itslowering action of human blood antibody titer concentration in a mouseto which human peripheral blood mononuclear cells (PBMC) aretransplanted are confirmed (Patent Document 2 and Non-Patent Documents 2and 3).

RELATED ART Patent Document

[Patent Document 1] WO 2012/018687

[Patent Document 2] WO 2008/150494

Non-Patent Document

[Non-Patent Document 1] Arthritis & Rheumatism (US) 2010; 62(7):1933-1943

[Non-Patent Document 2] Molecular Immunology (US) 2008; 45(15):3926-3933

[Non-Patent Document 3] The Journal of Immunology (US) 2011; 186(7):4223-4233

DISCLOSURE OF INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide an anti-human Igβantibody which crosslinks BCR and FcγRIIb and has an immunosuppressivefunction more enhanced than that of an antibody in the prior art.

Means for Solving the Problems

As a result of intensive research on preparation of an anti-human Igβantibody by the present inventors, a plurality of anti-human Igβantibodies comprising a heavy chain variable region comprising CDR1consisting of the amino acid sequence of amino acid numbers 31 to 35 ofSEQ ID NO: 2, CDR2 consisting of the amino acid sequence of amino acidnumbers 50 to 65 of SEQ ID NO: 2, and CDR3 consisting of the amino acidsequence of amino acid numbers 98 to 108 of SEQ ID NO: 2, and a lightchain variable region comprising CDR1 consisting of the amino acidsequence of amino acid numbers 24 to 38 of SEQ ID NO: 4, CDR2 consistingof the amino acid sequence of amino acid numbers 54 to 60 of SEQ ID NO:4, and CDR3 consisting of the amino acid sequence of amino acid numbers93 to 101 of SEQ ID NO: 4, in which a heavy chain constant region of theantibody is a human Igγ1 constant region having amino acid mutations ofS239D, H268D, and L328W were prepared (Examples 1 to 3), and it wasfound that these antibodies bind to human Igβ on human B cells (Examples4 and 5) and inhibit activation of the human B cells induced by ananti-IgM antibody (Example 6). As a result, the above-describedanti-human Igβ antibody is provided, thereby completing the presentinvention. Further, it was found that the antibody suppresses the plasmahuman antibody titer in a human PBMC transfer NOG mouse model (Example7) and suppresses an antigen-specific antibody without being affected bythe total antibody titers in plasma in a monkey TTx antigensensitization model (Example 8).

That is, the present invention includes the following invention as amaterial or a method which is medically or industrially applicable.

(1) An anti-human Igβ antibody comprising a heavy chain variable regioncomprising CDR1 consisting of the amino acid sequence of amino acidnumbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acidsequence of amino acid numbers 50 to 65 of SEQ ID NO: 2, and CDR3consisting of the amino acid sequence of amino acid numbers 98 to 108 ofSEQ ID NO: 2, a light chain variable region comprising CDR1 consistingof the amino acid sequence of amino acid numbers 24 to 38 of SEQ ID NO:4, CDR2 consisting of the amino acid sequence of amino acid numbers 54to 60 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence ofamino acid numbers 93 to 101 of SEQ ID NO: 4, and a heavy chain constantregion which is a human Igγ1 constant region having amino acid mutationsof S239D, H268D, and L328W.

(2) The anti-human Igβ antibody of (1) above which is a humanizedantibody.

(3) The anti-human Igβ antibody of (1) above, selected from the groupconsisting of the following 1) to 4):

1) an anti-human Igβ antibody comprising a heavy chain variable regionconsisting of the amino acid sequence of amino acid numbers 1 to 119 ofSEQ ID NO: 6, a light chain variable region consisting of the amino acidsequence of amino acid numbers 1 to 112 of SEQ ID NO: 8, and a heavychain constant region which is a human Igγ1 constant region having aminoacid mutations of S239D, H268D, and L328W;

2) an anti-human Igβ antibody comprising a heavy chain variable regionconsisting of the amino acid sequence of amino acid numbers 1 to 119 ofSEQ ID NO: 2, a light chain variable region consisting of the amino acidsequence of amino acid numbers 1 to 112 of SEQ ID NO: 4, and a heavychain constant region which is a human Igγ1 constant region having aminoacid mutations of S239D, H268D, and L328W;

3) an anti-human Igβ antibody comprising a heavy chain variable regionconsisting of the amino acid sequence of amino acid numbers 1 to 119 ofSEQ ID NO: 10, a light chain variable region consisting of the aminoacid sequence of amino acid numbers 1 to 112 of SEQ ID NO: 12, and aheavy chain constant region which is a human Igγ1 constant region havingamino acid mutations of S239D, H268D, and L328W; and

4) an anti-human Igβ antibody which is derived from posttranslationalmodification of the anti-human Igβ antibody of any one of (1) to (3)above.

(4) The anti-human Igβ antibody of (3) above, comprising a heavy chainvariable region consisting of the amino acid sequence of amino acidnumbers 1 to 119 of SEQ ID NO: 6, a light chain variable regionconsisting of the amino acid sequence of amino acid numbers 1 to 112 ofSEQ ID NO: 8, and a heavy chain constant region which is a human Igγ1constant region having amino acid mutations of S239D, H268D, and L328W.

(5) The anti-human Igβ antibody of (3) above, comprising a heavy chainvariable region consisting of the amino acid sequence of amino acidnumbers 1 to 119 of SEQ ID NO: 2, a light chain variable regionconsisting of the amino acid sequence of amino acid numbers 1 to 112 ofSEQ ID NO: 4, and a heavy chain constant region which is a human Igγ1constant region having amino acid mutations of S239D, H268D, and L328W.

(6) The anti-human Igβ antibody of (3) above, comprising a heavy chainvariable region consisting of the amino acid sequence of amino acidnumbers 1 to 119 of SEQ ID NO: 10, a light chain variable regionconsisting of the amino acid sequence of amino acid numbers 1 to 112 ofSEQ ID NO: 12, and a heavy chain constant region which is a human Igγ1constant region having amino acid mutations of S239D, H268D, and L328W.

(7) An anti-human Igβ antibody which is derived from posttranslationalmodification of the anti-human Igβ antibody of any one of (4) to (6)above.

(8) The anti-human Igβ antibody of (3) or (7) above, wherein theposttranslational modification is pyroglutamylation at the N terminal ofthe heavy chain variable region and/or deletion of lysine at the Cterminal of the heavy chain.

(9) The anti-human Igβ antibody of any one of (1) to (8) above,comprising a light chain constant region which is a human Igκ constantregion.

(10) The anti-human Igβ antibody of (1) above, comprising a heavy chainconsisting of the amino acid sequence shown by SEQ ID NO: 6 and a lightchain consisting of the amino acid sequence shown by SEQ ID NO: 8.

(11) The anti-human Igβ antibody of (1), comprising a heavy chainconsisting of the amino acid sequence shown by SEQ ID NO: 2 and a lightchain consisting of the amino acid sequence shown by SEQ ID NO: 4.

(12) The anti-human Igβ antibody of (1) above, comprising a heavy chainconsisting of the amino acid sequence shown by SEQ ID NO: 10 and a lightchain consisting of the amino acid sequence shown by SEQ ID NO: 12.

(13) An anti-human Igβ antibody which is derived from posttranslationalmodification of the anti-human Igβ antibody of any one of (10) to (12)above.

(14) The anti-human Igβ antibody of (13) above, wherein theposttranslational modification is pyroglutamylation at the N terminal ofthe heavy chain variable region and/or deletion of lysine at the Cterminal of the heavy chain.

(15) The anti-human Igβ antibody of (13) above, comprising a heavy chainconsisting of the amino acid sequence of amino acid numbers of 1 to 448of SEQ ID NO: 6 in which glutamine of amino acid number 1 is modified topyroglutamic acid and a light chain consisting of the amino acidsequence shown by SEQ ID NO:8.

(16) The anti-human Igβ antibody of (13) above, comprising a heavy chainconsisting of the amino acid sequence of amino acid numbers 1 to 448 ofSEQ ID NO:2 and a light chain consisting of the amino acid sequenceshown by SEQ ID NO:4.

(17) The anti-human Igβ antibody of (13) above, comprising a heavy chainconsisting of the amino acid sequence of amino acid numbers 1 to 448 ofSEQ ID NO:10 and a light chain consisting of the amino acid sequenceshown by SEQ ID NO:12.

(18) A polynucleotide comprising a base sequence encoding the heavychain of the anti-human Igβ antibody of any one of (1) to (6) above.

(19) A polynucleotide comprising a base sequence encoding the lightchain of the anti-human Igβ antibody of any one of (1) to (6) above.

(20) An expression vector comprising the polynucleotide of (18) and/or(19) above.

(21) A host cell transformed with the expression vector of (20) above,selected from the group consisting of the following (a) to (d):

(a) a host cell transformed with an expression vector comprising apolynucleotide comprising a base sequence encoding the heavy chain ofthe anti-human Igβ antibody of any one of (1) to (6) above and apolynucleotide comprising a base sequence encoding the light chain ofthe antibody;

(b) a host cell transformed with an expression vector comprising apolynucleotide comprising a base sequence encoding the heavy chain ofthe anti-human Igβ antibody of any one of (1) to (6) above and anexpression vector comprising a polynucleotide comprising a base sequenceencoding the light chain of the antibody;

(c) a host cell transformed with an expression vector comprising apolynucleotide comprising a base sequence encoding the heavy chain ofthe anti-human Igβ antibody of any one of (1) to (6) above; and

(d) a host cell transformed with an expression vector comprising apolynucleotide comprising a base sequence encoding the light chain ofthe anti-human Igβ antibody of any one of (1) to (6) above.

(22) A host cell transformed with the expression vector of (20) above,selected from the group consisting of the following (a) to (d):

(a) a host cell transformed with an expression vector comprising apolynucleotide comprising a base sequence encoding the heavy chain ofthe anti-human Igβ antibody of any one of (10) to (13) above and apolynucleotide comprising a base sequence encoding the light chain ofthe antibody;

(b) a host cell transformed with an expression vector comprising apolynucleotide comprising a base sequence encoding the heavy chain ofthe anti-human Igβ antibody of any one of (10) to (13) above and anexpression vector comprising a polynucleotide comprising a base sequenceencoding the light chain of the antibody;

(c) a host cell transformed with an expression vector comprising apolynucleotide comprising a base sequence encoding the heavy chain ofthe anti-human Igβ antibody of any one of (10) to (13) above; and

(d) a host cell transformed with an expression vector comprising apolynucleotide comprising a base sequence encoding the light chain ofthe anti-human Igβ antibody of any one of (10) to (13) above.

(23) A method for producing an anti-human Igβ antibody comprisingculturing host cell(s) selected from the group consisting of thefollowing (a) to (c) to express the anti-human Igβ antibody:

(a) a host cell transformed with an expression vector comprising apolynucleotide comprising a base sequence encoding the heavy chain ofthe anti-human Igβ antibody of any one of (1) to (6) above and apolynucleotide comprising a base sequence encoding the light chain ofthe antibody;

(b) a host cell transformed with an expression vector comprising apolynucleotide comprising a base sequence encoding the heavy chain ofthe anti-human Igβ antibody of any one of (1) to (6) above and anexpression vector comprising a polynucleotide comprising a base sequenceencoding the light chain of the antibody; and

(c) a host cell transformed with an expression vector comprising apolynucleotide comprising a base sequence encoding the heavy chain ofthe anti-human Igβ antibody of any one of (1) to (6) above and a hostcell transformed with an expression vector comprising a polynucleotidecomprising a base sequence encoding the light chain of the antibody.

(24) A method for producing an anti-human Igβ antibody comprisingculturing host cell(s) selected from the group consisting of thefollowing (a) to (c) to express the anti-human Igβ antibody:

(a) a host cell transformed with an expression vector comprising apolynucleotide comprising a base sequence encoding the heavy chain ofthe anti-human Igβ antibody of any one of (10) to (13) above and apolynucleotide comprising a base sequence encoding the light chain ofthe antibody;

(b) a host cell transformed with an expression vector comprising apolynucleotide comprising a base sequence encoding the heavy chain ofthe anti-human Igβ antibody of any one of (10) to (13) above and anexpression vector comprising a polynucleotide comprising a base sequenceencoding the light chain of the antibody; and

(c) a host cell transformed with an expression vector comprising apolynucleotide comprising a base sequence encoding the heavy chain ofthe anti-human Igβ antibody of any one of (10) to (13) above and a hostcell transformed with an expression vector comprising a polynucleotidecomprising a base sequence encoding the light chain of the antibody.

(25) An anti-human Igβ antibody which is produced by the method of (23)above.

(26) An anti-human Igβ antibody which is produced by the method of (24)above.

(27) A pharmaceutical composition comprising the anti-human Igβ antibodyof any one of (1) to (17), (25), and (26) above and a pharmaceuticallyacceptable excipient.

(28) A pharmaceutical composition comprising the anti-human Igβ antibodyof (10) above, the anti-human Igβ antibody of (15) above, and apharmaceutically acceptable excipient.

(29) The pharmaceutical composition of (27) or (28) above, which is apharmaceutical composition for preventing or treating an autoimmunedisease.

(30) The pharmaceutical composition of (29) above, wherein theautoimmune disease is systemic lupus erythematosus, rheumatoidarthritis, or idiopathic thrombocytopenic purpura.

(31) A method for preventing or treating an autoimmune disease,comprising administrating a therapeutically effective amount of theanti-human Igβ antibody of any one of (1) to (17), (25), and (26) above.

(32) The method of (1) to (17), (25), and (26) above, wherein theautoimmune disease is systemic lupus erythematosus, rheumatoidarthritis, or idiopathic thrombocytopenic purpura.

(33) The anti-human Igβ antibody of any one of (1) to (17), (25), and(26) above for use in preventing or treating an autoimmune disease.

(34) The anti-human Igβ antibody of (33) above, wherein the autoimmunedisease is systemic lupus erythematosus, rheumatoid arthritis, oridiopathic thrombocytopenic purpura.

(35) Use of the anti-human Igβ antibody of any one of (1) to (17), (25),and (26) above for manufacture of a pharmaceutical composition forpreventing or treating an autoimmune disease.

(36) The use of (35) above, wherein the autoimmune disease is systemiclupus erythematosus, rheumatoid arthritis, or idiopathicthrombocytopenic purpura.

Effects of the Invention

An anti-human Igβ of the present invention has an excellentimmunosuppressive action by means of inhibiting activation of B cellsand can be used as an agent for preventing or treating of autoimmunediseases such as systemic lupus erythematosus, rheumatoid arthritis, andidiopathic thrombocytopenic purpura.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an inhibitory effect of a humanized anti-Igβ antibodyagainst anti-IgM antibody-induced cell proliferation in human B cells.The vertical axis indicates a rate of proliferation of B cells and thehorizontal axis indicates added antibody concentration (μg/mL).

FIG. 2 shows an inhibitory action of a humanized anti-Igβ antibodyagainst an increase in human IgM antibody titers in plasma induced bytransfer of human PBMC into an NOG mouse. The vertical axis indicatesthe human IgM antibody titer in plasma (μg/mL) and the horizontal axisindicates the time (day) from transferring the human PBMC into the NOGmouse.

FIG. 3 shows an inhibitory action of a humanized anti-Igβ antibodyagainst an increase in human IgE antibody titers in plasma induced bytransfer of human PBMC into an NOG mouse. The vertical axis indicatesthe human IgE antibody titer in plasma (ng/mL) and the horizontal axisindicates the time (day) from transferring the human PBMC into the NOGmouse.

FIG. 4 shows an inhibitory action of a humanized anti-Igβ antibodyagainst an increase in anti-adsorbed tetanus toxoid in plasma caused byimmunizing adsorbed tetanus toxoid to a monkey. The vertical axisindicates the anti-adsorbed tetanus toxoid antibody titer in plasma(U/mL) and the horizontal axis indicates the time (day) from immunizingadsorbed tetanus toxoid to a monkey.

FIG. 5 shows an action of the humanized anti-Igβ antibody against totalIgM antibody titer in plasma of the monkey immunized by adsorbed tetanustoxoid. The vertical axis indicates the total IgM antibody titer inplasma (U/mL) and the horizontal axis indicates the time (day) fromimmunizing adsorbed tetanus toxoid to a monkey.

FIG. 6 shows an action of the humanized anti-Igβ antibody against totalIgA antibody titer in plasma of the monkey immunized by adsorbed tetanustoxoid. The vertical axis indicates the total IgA antibody titer inplasma (U/mL) and the horizontal axis indicates the time (day) fromimmunizing adsorbed tetanus toxoid to a monkey.

FIG. 7 shows an action of the humanized anti-Igβ antibody against totalIgG antibody titer in plasma of the monkey immunized by adsorbed tetanustoxoid. The vertical axis indicates the total IgG antibody titer inplasma (U/mL) and the horizontal axis indicates the time (day) fromimmunizing adsorbed tetanus toxoid to a monkey.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

There are five classes of IgG, IgM, IgA, IgD, and IgE in an antibody.The basic structure of an antibody molecule is configured of heavychains having a molecular weight of 50000 to 70000 and light chainshaving a molecular weight of 20000 to 30000 in each of the classes incommon. Heavy chain usually consists of a polypeptide chain comprisingapproximately 440 amino acids, has a distinctive structure for each ofthe classes, and is referred to as Igγ, Igμ, Igα, Igδ, and Igεcorresponding to IgG, IgM, IgA, IgD, and IgE, respectively. Further,four subclasses of IgG1, IgG2, IgG3, and IgG4 are present in IgG and theheavy chains respectively corresponding thereto are referred to as Igγ1,Igγ2, Igγ3, and Igγ4. Light chain usually consists of a polypeptidechain comprising 220 amino acids, two types of which, type L and type Kare known, and are referred to as Igδ and Igκ. In a peptideconfiguration of the basic structure of antibody molecules, twohomologous heavy chains and two homologous light chains are bound bydisulfide bonds (S—S bond) and non-covalent bonds, and the molecularweight thereof is 150000 to 190000. Two kinds of light chains can bepaired with any heavy chain. The respective antibody molecules typicallyconsist of two identical light chains and two identical heavy chains.

With regard to intrachain S—S bonds, four of the S—S bonds are presentin the heavy chain (five in μ and ε chains) and two of them are presentin the light chain; one loop is formed per 100 to 110 amino acidresidues, and this steric structure is similar among the loops and arereferred to as a structural unit or a domain. The domain located at theamino terminal side (N terminal side) in both of the heavy chain and thelight chain, whose amino acid sequence is not constant even in a case ofa sample from the same class (sub class) of the same kind of animal isreferred to as a variable region, and respective domains are referred toas a heavy chain variable region and a light chain variable region. Theamino acid sequence of the carboxy terminal side (C terminal side) fromthe variable region is nearly constant in each class or subclass and isreferred to as a constant region.

An antigenic binding site of an antibody is configured of the heavychain variable region and the light chain variable region, and thebinding specificity depends on the amino acid sequence of this site. Onthe other hand, biological activities such as binding to complements andvarious cells reflect differences in the constant region structuresamong each class Ig. It is understood that the variability of variableregions of the light chains and the heavy chains is mostly limited tothree small hypervariable regions present in both chains and theseregions are referred to as complementarity determining regions (CDR:CDR1, CDR2, and CDR3 from the N terminal side). The remaining portion ofthe variable region is referred to as a framework region (FR) and isrelatively constant.

<Anti-Human Igβ Antibody of the Present Invention>

The anti-human Igβ antibody of the present invention includes ananti-human Igβ antibody having the following characteristics.

An anti-human Igβ antibody comprising a heavy chain variable regioncomprising CDR1 consisting of the amino acid sequence of amino acidnumbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acidsequence of amino acid numbers 50 to 65 of SEQ ID NO: 2, and CDR3consisting of the amino acid sequence of amino acid numbers 98 to 108 ofSEQ ID NO: 2, a light chain variable region comprising CDR1 consistingof the amino acid sequence of amino acid numbers 24 to 38 of SEQ ID NO:4, CDR2 consisting of the amino acid sequence of amino acid numbers 54to 60 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence ofamino acid numbers 93 to 101 of SEQ ID NO: 4, and a heavy chain constantregion which is a human Igγ1 constant region having amino acid mutationsof S239D, H268D, and L328W.

In one embodiment, the anti-human Igβ antibody of the present inventionis a humanized antibody. The “humanized antibody” in the presentspecification means an antibody in a form comprising CDRs derived from amouse antibody and other antibody portions derived from a humanantibody. A method for preparing a humanized antibody is a known in theart and can be prepared with reference to U.S. Pat. Nos. 5,225,539,6,180,370, and the like.

In one embodiment, the anti-human Igβ antibody of the present inventionis an anti-human Igβ antibody described in any one of the following 1)to 3):

1) an anti-human Igβ antibody comprising a heavy chain variable regionconsisting of the amino acid sequence of amino acid numbers 1 to 119 ofSEQ ID NO: 2, a light chain variable region consisting of the amino acidsequence of amino acid numbers 1 to 112 of SEQ ID NO: 4, and a heavychain constant region which is a human Igγ1 constant region having aminoacid mutations of S239D, H268D, and L328W;

2) an anti-human Igβ antibody comprising a heavy chain variable regionconsisting of the amino acid sequence of amino acid numbers 1 to 119 ofSEQ ID NO: 6, a light chain variable region consisting of the amino acidsequence of amino acid numbers 1 to 112 of SEQ ID NO: 8, and a heavychain constant region which is a human Igγ1 constant region having aminoacid mutations of S239D, H268D, and L328W; and

3) an anti-human Igβ antibody comprising a heavy chain variable regionconsisting of the amino acid sequence of amino acid numbers 1 to 119 ofSEQ ID NO: 10, a light chain variable region consisting of the aminoacid sequence of amino acid numbers 1 to 112 of SEQ ID NO: 12, and aheavy chain constant region which is a human Igγ1 constant region havingamino acid mutations of S239D, H268D, and L328W.

The number of residue regarding introduction of amino acid mutations inan antibody constant region used in the present specification followsthe EU index (Kabat et al. 1991, Sequences of Proteins of ImmunologicalInterest, 5^(th) Ed., United States Public Health Service, NationalInstitute of Health, Bethesda). S239D is replacement of serine at239^(th) position of the amino acid according to the EU index of Kabatet al. in the human Igγ1 constant region with aspartic acid. H268D isreplacement of histidine at 268^(th) position of the amino acidaccording to the EU index of Kabat et al. in the human Igγ1 constantregion with aspartic acid. L328W is replacement of leucine at 328^(th)position of the amino acid according to the EU index of Kabat et al. inthe human Igγ1 constant region with triptophan. Examples of the humanIgγ1 constant region having amino acid mutations of S239D, H268D, andL328W include a human Igγ1 constant region consisting of the amino acidsequence of amino acid numbers 120 to 449 of SEQ ID NO: 2.

As the light chain constant region of the anti-human Igβ antibody of thepresent invention, any one of constant region of Igλ and Igκ can beselected, but a human Igκ constant region is preferable. Examples of thehuman Igκ constant region include a human Igκ constant region consistingof amino acid sequence of amino acid numbers 113 to 218 of SEQ ID NO: 4.

In one embodiment, the anti-human Igβ antibody of the present inventionis an anti-human Igβ antibody selected from any one of the following i)to iii):

i) an anti-human Igβ antibody comprising a heavy chain consisting of theamino acid sequence shown by SEQ ID NO: 2 and a light chain consistingof the amino acid sequence shown by SEQ ID NO: 4;

ii) an anti-human Igβ antibody comprising a heavy chain consisting ofthe amino acid sequence shown by SEQ ID NO: 6 and a light chainconsisting of the amino acid sequence shown by SEQ ID NO: 8; and

iii) an anti-human Igβ antibody comprising a heavy chain consisting ofthe amino acid sequence shown by SEQ ID NO: 10 and a light chainconsisting of the amino acid sequence shown by SEQ ID NO: 12.

It is known that when an antibody is expressed in cells, the antibody ismodified after translation. Examples of the posttranslationalmodification include cleavage of lysine at the C terminal of the heavychain by a carboxypeptidase; modification of glutamine or glutamic acidat the N terminal of the heavy chain and the light chain to pyroglutamicacid by pyroglutamylation; glycosylation; oxidation; deamidation; andglycation, and it is known that such posttranslational modificationsoccur in various antibodies (Journal of Pharmaceutical Sciences, Vol.97, p. 2426-2447, 2008).

The anti-human Igβ antibody of the present invention includes ananti-human Igβ antibody which has undergone posttranslationalmodification. Examples of the anti-human Igβ antibody of the presentinvention which undergoes posttranslational modification includeanti-human Igβ antibodies which have undergone pyroglutamylation at theN terminal of the heavy chain variable region and/or deletion of lysineat the C terminal of the heavy chain. It is known in the field that suchposttranslational modification due to pyroglutamylation at the Nterminal and deletion of lysine at the C terminal does not have anyinfluence on the activity of the antibody (Analytical Biochemistry, Vol.348, p. 24-39, 2006).

For example, the anti-human Igβ antibodies of the present inventioninclude an anti-human Igβ antibody described in any one of thefollowing 1) to 3):

1) an anti-human Igβ antibody comprising a heavy chain consisting of theamino acid sequence of SEQ ID NO: 2 in which glutamic acid of amino acidnumber 1 is modified to pyroglutamic acid and/or lysine of amino acidnumber 449 is deleted and a light chain consisting of the amino acidsequence shown by SEQ ID NO: 4;

2) an anti-human Igβ antibody comprising a heavy chain consisting of theamino acid sequence of SEQ ID NO: 6 in which glutamic acid of amino acidnumber 1 is modified to pyroglutamic acid and/or lysine of amino acidnumber 449 is deleted and a light chain consisting of the amino acidsequence shown by SEQ ID NO: 8; and

3) an anti-human Igβ antibody comprising a heavy chain consisting of theamino acid sequence of SEQ ID NO: 10 in which glutamic acid of aminoacid number 1 is modified to pyroglutamic acid and/or lysine of aminoacid number 449 is deleted and a light chain consisting of the aminoacid sequence shown by SEQ ID NO: 12.

In one embodiment, the anti-human Igβ antibody of the present inventionis an anti-human Igβ antibody selected from any one of the following i)to iii):

i) an anti-human Igβ antibody comprising a heavy chain consisting of theamino acid sequence of amino acid numbers of 1 to 448 of SEQ ID NO: 2and a light chain consisting of the amino acid sequence shown by SEQ IDNO: 4;

ii) an anti-human Igβ antibody comprising a heavy chain consisting ofthe amino acid sequence of amino acid numbers 1 to 448 of SEQ ID NO: 6in which glutamine of amino acid number 1 is modified to pyroglutamicacid and a light chain consisting of the amino acid sequence shown bySEQ ID NO: 8; and

iii) an anti-human Igβ antibody comprising a heavy chain consisting ofthe amino acid sequence of amino acid numbers 1 to 448 of SEQ ID NO: 10and a light chain consisting of the amino acid sequence shown by SEQ IDNO: 12.

Any person skilled in the art can prepare a fused form of an antibodyand another peptide or protein and can also prepare a modified form towhich a modifying agent binds on the basis of the present invention, andthe antibody of the present invention includes the antibody in theseforms. Other peptides or proteins used for the fusion is notparticularly limited as long as the binding activity of the antibody isnot decreased, and examples thereof include human serum albumin, varioustag peptides, artificial helix motif peptide, maltose-binding proteins,glutathione S transferase, various toxins, other peptides or proteinscapable of promoting multimerization, and the like. The modifying agentused for the modification is not particularly limited as long as thebinding activity of the antibody is not decreased, and examples thereofinclude polyethylene glycol, sugar chains, phospholipids, liposomes,low-molecular compounds, and the like.

The “anti-human Igβ antibody” in the present specification means anantibody binding to a human Igβ. Whether the “anti-human Igβ antibody”binds to a human Igβ is confirmed by using a known binding activitymeasurement method. Examples of the binding activity measurement methodinclude a method of Enzyme-Linked ImmunoSorbent Assay (ELISA) and thelike. In a case of using the ELISA, for example, human Igβ-Flag protein(for example, encoded by the base sequence of SEQ ID NO: 13) issolidified on the ELISA Plate and a test antibody is added thereto to bereacted. After the reaction, a secondary antibody such as an anti-IgGantibody, labeled with an enzyme such as horseradish peroxidase (HRP) orthe like, is reacted, and washed off, and then it is possible to confirmwhether the test antibody binds to the human Igβ by identifying bindingof the secondary antibody through activity measurement using a reagentdetecting the activity (for example, in a case of HRP labeling,BM-Chemiluminescence ELISA Substrate (POD) (Roche Diagnostics Inc.)). Asa specific measurement method, the method described in Example 4 belowcan be used.

The anti-human Igβ antibody of the present invention includes, inaddition to binding to human Igβ, an antibody binding to Igβ derivedfrom other animals (for example, monkey Igβ), as long as the antibodybinds to human Igβ.

As a method for evaluating the activity of the anti-human Igβ antibodyof the present invention, the binding activity on human B cells or theactivity of inhibiting activation of the human B cells induced by BCRstimulation may be evaluated. As the methods of evaluating suchactivity, the methods described in Examples 5 and 6 below can be used.Preferably, the anti-human Igβ antibody of the present invention has anactivity of binding to human Igβ and inhibiting activation of human Bcells induced by BCR stimulation.

The anti-human Igβ antibody of the present invention can be easilyprepared by a person skilled in the art using a known method in thefield, based on sequence information on the heavy chain and the lightchain of the antibody of the present invention, which is disclosed inthe present specification. The anti-human Igβ antibody of the presentinvention is not particularly limited, but can be produced according tothe method described in the section of <Method of producing anti-humanIgβ antibody of the present invention, and anti-human Igβ antibodyproduced by the method> described below.

The anti-human Igβ antibody of the present invention is further purifiedas needed, formulated according to a conventional method, and may beused for the prevention or the treatment of autoimmune diseases such assystemic lupus erythematosus, rheumatoid arthritis, idiopathicthrombocytopenic purpura, myasthenia gravis, Grave's disease, opticneuromyelitis, autoimmune hemolytic anemia, pemphigus, antiphospholipidantibody syndrome, ANCA associated vasculitis, Sjogren's syndrome,Hashimoto's disease, chronic inflammatory demyelinating polyneuropathy,or chronic fatigue syndrome.

<Polynucleotide of the Present Invention>

The polynucleotide of the present invention includes a polynucleotidecomprising a base sequence encoding the heavy chain of the anti-humanIgβ antibody of the present invention and a polynucleotide comprising abase sequence encoding the light chain of the anti-human Igβ antibody ofthe present invention.

In one embodiment, the polynucleotide comprising a base sequenceencoding the heavy chain of the anti-human Igβ antibody of the presentinvention is a polynucleotide comprising a base sequence encoding theheavy chain consisting of the amino acid sequence shown by SEQ ID NO: 2,a polynucleotide comprising a base sequence encoding the heavy chainconsisting of the amino acid sequence shown by SEQ ID NO: 6, or apolynucleotide comprising a base sequence encoding the heavy chainconsisting of the amino acid sequence shown by SEQ ID NO: 10.

Examples of the polynucleotide comprising a base sequence encoding theheavy chain consisting of the amino acid sequence shown by SEQ ID NO: 2include a polynucleotide comprising the base sequence shown by SEQ IDNO: 1 or 15. Examples of the polynucleotide comprising a base sequenceencoding the heavy chain consisting of the amino acid sequence shown bySEQ ID NO: 6 include a polynucleotide comprising the base sequence shownby SEQ ID NO: 5. Examples of the polynucleotide comprising a basesequence encoding the heavy chain consisting of the amino acid sequenceshown by SEQ ID NO: 10 include a polynucleotide comprising the basesequence shown by SEQ ID NO: 9.

In one embodiment, the polynucleotide comprising a base sequenceencoding the light chain of the anti-human Igβ antibody of the presentinvention is a polynucleotide comprising a base sequence encoding thelight chain consisting of the amino acid sequence shown by SEQ ID NO: 4,a polynucleotide comprising a base sequence encoding the light chainconsisting of the amino acid sequence shown by SEQ ID NO: 8, or apolynucleotide comprising a base sequence encoding the light chainconsisting of the amino acid sequence shown by SEQ ID NO: 12.

Examples of the polynucleotide comprising a base sequence encoding thelight chain consisting of the amino acid sequence shown by SEQ ID NO: 4include a polynucleotide comprising the base sequence shown by SEQ IDNO: 3. Examples of the polynucleotide comprising a base sequenceencoding the light chain consisting of the amino acid sequence shown bySEQ ID NO: 8 include a polynucleotide comprising the base sequence shownby SEQ ID NO: 7. Examples of the polynucleotide comprising a basesequence encoding the light chain consisting of the amino acid sequenceshown by SEQ ID NO: 12 include a polynucleotide comprising the basesequence shown by SEQ ID NO: 11.

The polynucleotide of the present invention can be easily prepared by aperson skilled in the art using a known method in the field based on thebase sequence. For example, the polynucleotide of the present inventioncan be synthesized using a known gene synthesis method in the field. Asthe gene synthesis method, various methods such as a synthesis method ofantibody genes described in WO90/07861 known by a person skilled in theart can be used.

<Expression Vector of the Present Invention>

An expression vector of the present invention includes an expressionvector comprising a polynucleotide comprising a base sequence encodingthe heavy chain of the anti-human Igβ antibody of the present invention,an expression vector comprising a polynucleotide comprising a basesequence encoding the light chain of the anti-human Igβ antibody of thepresent invention, and an expression vector comprising a polynucleotidecomprising a base sequence encoding the heavy chain of the anti-humanIgβ antibody of the present invention and a polynucleotide comprising abase sequence encoding the light chain of the antibody.

The expression vector used to express the polynucleotide of the presentinvention are not particularly limited as long as a polynucleotidecomprising the base sequence encoding the heavy chain of the anti-humanIgβ antibody of the present invention and/or a polynucleotide comprisingthe base sequence encoding the light chain of the anti-human Igβantibody of the present invention can be expressed in various host cellsof eukaryotic cells (for example, animal cells, insect cells, plantcells, and yeast) and/or prokaryotic cells (for example, Escherichiacoli), and the polypeptides encoded by these can be produced. Examplesof the expression vector include plasmid vectors, viral vectors (forexample, adenovirus or retrovirus), and the like. Preferably pEE6.4 orpEE02.4 (Lonza Biologics, Inc.) can be used.

The expression vector of the present invention may include a promoterthat is operably linked to the polynucleotide of the present invention.Examples of the promoter for expressing the polynucleotide of theinvention with animal cells include a virus-derived promoter such asCMV, RSV, or SV40, an actin promoter, an EF (elongation factor) 1αpromoter, and a heat shock promoter. Examples of promoters forexpressing the polynucleotide of the invention by bacteria (for example,Escherichia) include a trp promoter, a lac promoter, XPL promoter, andtac promoter. Further, examples of promoters for expressing thepolynucleotide of the invention by yeast include a GAL 1 promoter, a GAL10 promoter, a PH05 promoter, a PGK promoter, a GAP promoter, and an ADHpromoter.

In the case of using an animal cell, an insect cell, or yeast as thehost cell, the expression vector of the present invention may compriseinitiation codon and termination codon. In this case, the expressionvector of the present invention may comprise an enhancer sequence, anuntranslated region on the 5′ side and the 3′ side of genes encoding theantibody of the present invention or the heavy chain or the light chain,a secretory signal sequence, a splicing junction, a polyadenylationsite, or a replicable unit. When Escherichia coli is used as the hostcell, the expression vector of the present invention may comprise aninitiation codon, a termination codon, a terminator region, and areplicable unit. In this case, the expression vector of the presentinvention may comprise a selection marker (for example, tetracyclineresistant genes, ampicillin resistant genes, kanamycin resistant genes,neomycin resistant genes, or dihydrofolate reductase genes) which isgenerally used according to the necessity.

<Transformed Host Cell of the Present Invention>

The transformed host cell of the present invention includes a host celltransformed with the expression vector of the present invention, whichis selected from the group consisting of the following (a) to (d):

(a) a host cell transformed with an expression vector comprising apolynucleotide comprising a base sequence encoding the heavy chain ofthe anti-human Igβ antibody of the present invention and apolynucleotide comprising a base sequence encoding the light chain ofthe antibody;

(b) a host cell transformed with an expression vector comprising apolynucleotide comprising a base sequence encoding the heavy chain ofthe anti-human Igβ antibody of the present invention and an expressionvector comprising a polynucleotide comprising a base sequence encodingthe light chain of the antibody;

(c) a host cell transformed with an expression vector comprising apolynucleotide comprising a base sequence encoding the heavy chain ofthe anti-human Igβ antibody of the present invention; and

(d) a host cell transformed with an expression vector comprising apolynucleotide comprising a base sequence encoding the light chain ofthe anti-human Igβ antibody of the present invention.

Examples of the preferred transformed host cell of the present inventioninclude a host cell transformed with an expression vector comprising apolynucleotide comprising a base sequence encoding the heavy chain ofthe anti-human Igβ antibody of the present invention and apolynucleotide comprising a base sequence encoding the light chain ofthe antibody, and a host cell transformed with an expression vectorcomprising a polynucleotide comprising a base sequence encoding theheavy chain of the anti-human Igβ antibody of the present invention andan expression vector comprising a polynucleotide comprising a basesequence encoding the light chain of the antibody.

The transformed host cell is not particularly limited as long as thehost cell is appropriate for the expression vector being used,transformed with the expression vector, and can express the antibody.Examples of the transformed host cell include various cells such asnatural cells or artificially established cells which are generally usedin the field of the present invention (for example, animal cells (forexample, CHO-K1SV cells), insect cells (for example, Sf9), bacteria (forexample, Escherichia), yeast (for example, Saccharomyces or Pichia) orthe like). Preferably cultured cells such as CHO-K1SV cells, CHO-DG 44cells, 293 cells, or NS0 cells can be used.

A method of transforming the host cell is not particularly limited, but,for example, a calcium phosphate method or an electroporation method canbe used.

<Method of Producing Anti-Human Igβ Antibody of the Present Invention,and Anti-Human Igβ Antibody Produced by the Method>

The method for producing the anti-human Igβ antibody of the presentinvention include a method for producing an anti-human Igβ antibody byculturing host cell(s) selected from the group consisting of thefollowing (a) to (c) to express the anti-human Igβ antibody:

(a) a host cell transformed with an expression vector comprising apolynucleotide comprising a base sequence encoding the heavy chain ofthe anti-human Igβ antibody of the present invention and apolynucleotide comprising a base sequence encoding the light chain ofthe antibody;

(b) a host cell transformed with an expression vector comprising apolynucleotide comprising a base sequence encoding the heavy chain ofthe anti-human Igβ antibody of the present invention and an expressionvector comprising a polynucleotide comprising a base sequence encodingthe light chain of the antibody; and

(c) a host cell transformed with an expression vector comprising apolynucleotide comprising a base sequence encoding the heavy chain ofthe anti-human Igβ antibody of the present invention and a host celltransformed with an expression vector comprising a polynucleotidecomprising a base sequence encoding the light chain of the antibody.

The method for producing the anti-human Igβ antibody of the presentinvention is not particularly limited as long as it includes a step ofculturing the transformed host cells of the present invention to expressthe anti-human Igβ antibody. Examples of the preferred host cells usedin the method include the preferred transformed host cells of thepresent invention as described above.

The transformed host cell can be cultured by known methods. Cultureconditions, for example, the temperature, pH of culture medium, and theculture time are appropriately selected. In a case where the host cellis an animal cell, examples of the culture medium include MEM culturemedium supplemented with approximately 5% to 20% of fetal bovine serum(Science, Vol. 130, p. 432-437, 1959), DMEM culture medium (Virology,Vol. 8, p. 396, 1959), RPMI1640 culture medium (J. Am. Med. Assoc., Vol.199, p. 519, 1967), and a 199 culture medium (Exp. Biol. Med., Vol. 73,p. 1-8, 1950). The pH of the culture medium is preferably approximately6 to 8, and the culture is generally carried out at approximately 30° C.to 40° C. for approximately 15 hours to 72 hours while air ventilatingand stirring if necessary. In a case where the host cell is an insectcell, as the culture medium, for example, Grace's culture medium (Proc.Natl. Acad. Sci. USA, Vol. 82, p. 8404, 1985) supplemented with fetalbovine serum can be used. The pH of the culture medium is preferablyapproximately 5 to 8, and the culture is generally carried out atapproximately 20° C. to 40° C. for approximately 15 hours to 100 hourswhile air ventilating and stirring if necessary. In a case where thehost cell is Escherichia coli or yeast, as the culture medium, forexample, liquid culture medium supplemented with a source of nutrientsis appropriate. It is preferable that the nutrient culture mediuminclude a carbon source, an inorganic nitrogen source, or an organicnitrogen source necessary for the growth of the transformed host cell.Examples of the carbon source include glucose, dextran, soluble starch,and sucrose and examples of the inorganic nitrogen source or the organicnitrogen source include ammonium salts, nitrate salts, amino acids, cornsteep liquor, peptone, casein, meat extract, soybean meal, and potatoextract. Other nutrients (for example, inorganic salts (for example,calcium chloride, sodium dihydrogen phosphate, and magnesium chloride),vitamins), and antibiotics (for example, tetracycline, neomycin,ampicillin, and kanamycin) may be included as desired. The pH of theculture medium is preferably approximately 5 to 8. In a case where thehost cell is Escherichia coli, preferred examples of the culture mediuminclude LB culture medium and M9 culture medium (Mol. Clo., Cold SpringHarbor Laboratory, Vol. 3, A2.2). The culture is generally carried outat approximately 14° C. to 43° C. for approximately 3 hours to 24 hourswhile air ventilating and stirring if necessary. In a case where thehost cell is yeast, as the culture medium, for example, Burkholderminimal medium (Proc. Natl. Acad, Sci, USA, Vol. 77, p. 4505, 1980) canbe used. The culture is generally carried out at approximately 20° C. to35° C. for approximately 14 hours to 144 hours while air ventilating andstirring if necessary. By carrying out the culture in theabove-described manner, it is possible to express the anti-human Igβantibody of the present invention.

The method of producing the anti-human Igβ antibody of the presentinvention may include recovering, preferably isolating or purifying theanti-human Igβ antibody from the transformed host cell in addition toculturing the transformed host cell of the present invention to expressthe anti-human Igβ antibody. Examples of the isolation or purificationmethod include methods using solubility such as salting-out and thesolvent precipitation method, methods using the difference in molecularweight such as dialysis, ultrafiltration, and gel filtration, methodsusing an electric charge such as ion exchange chromatography andhydroxylapatite chromatography, methods using specific affinity such asaffinity chromatography, methods using the difference in hydrophobicitysuch as reverse phase high performance liquid chromatography, andmethods using the difference in the isoelectric point such asisoelectric focusing phoresis. Preferably, the antibody accumulated in aculture supernatant can be purified by various chromatographies, forexample, column chromatography using Protein A column or Protein Gcolumn.

The anti-human Igβ antibody of the present invention also includes ananti-human Igβ antibody produced by the method for producing theanti-human Igβ antibody of the present invention.

<Pharmaceutical Composition of the Present Invention>

The pharmaceutical compositions of the present invention include apharmaceutical composition comprising the anti-human Igβ antibody of thepresent invention and pharmaceutically acceptable excipients. Thepharmaceutical composition of the present invention can be prepared by amethod being generally used with excipients, that is, excipients formedicine or carriers for medicine being generally used in the field.Examples of dosage forms of the pharmaceutical compositions includeparenteral drug such as an injection drug and a drip infusion drug, andthese can be administered by intravenous administration, subcutaneousadministration, or the like. In drug preparation, excipients, carriers,and additives in accordance with the dosage forms can be used within thepharmaceutically acceptable range.

The pharmaceutical compositions of the present invention may includeplural kinds of anti-human Igβ antibody of the present invention. Forexample, the present invention includes a pharmaceutical compositioncomprising an antibody which does not undergo posttranslationalmodification and an antibody derived from posttranslational modificationof the antibody.

In one embodiment, the pharmaceutical composition of the presentinvention comprises an anti-human Igβ antibody selected from the groupconsisting of the following (1) to (3) and an anti-human Igβ antibodyderived from posttranslational modification of the anti-human Igβantibody:

(1) an anti-human Igβ antibody comprising a heavy chain variable regionconsisting of the amino acid sequence of amino acid numbers 1 to 119 ofSEQ ID NO: 6, a light chain variable region consisting of the amino acidsequence of amino acid numbers 1 to 112 of SEQ ID NO: 8, and a heavychain constant region which is a human Igγ1 constant region having aminoacid mutations of S239D, H268D, and L328W;

(2) an anti-human Igβ antibody comprising a heavy chain variable regionconsisting of the amino acid sequence of amino acid numbers 1 to 119 ofSEQ ID NO: 2, a light chain variable region consisting of the amino acidsequence of amino acid numbers 1 to 112 of SEQ ID NO: 4, and a heavychain constant region which is a human Igγ1 constant region having aminoacid mutations of S239D, H268D, and L328W; and

(3) an anti-human Igβ antibody comprising a heavy chain variable regionconsisting of the amino acid sequence of amino acid numbers 1 to 119 ofSEQ ID NO: 10, a light chain variable region consisting of the aminoacid sequence of amino acid numbers 1 to 112 of SEQ ID NO: 12, and aheavy chain constant region which is a human Igγ1 constant region havingamino acid mutations of S239D, H268D, and L328W.

The pharmaceutical compositions of the present invention include apharmaceutical composition comprising an antibody in which lysine at theC terminal of the heavy chain is deleted, an antibody which hasundergone post-translational modification to the N terminal, an antibodyin which lysine at the C terminal of the heavy chain is deleted andwhich has undergone post-translation modification to N terminal, and/oran antibody which has lysine at the C terminal of the heavy chain anddoes not undergo post-translational modification to the N terminal.

In one embodiment, the pharmaceutical composition of the presentinvention comprising an anti-human Igβ antibody includes apharmaceutical composition comprising two or more anti-human Igβantibodies selected from the following (1) to (4):

(1) an anti-human Igβ antibody comprising a heavy chain consisting ofthe amino acid sequence of amino acid numbers 1 to 448 of SEQ ID NO: 2and a light chain consisting of the amino acid sequence shown by SEQ IDNO: 4;

(2) an anti-human Igβ antibody comprising a heavy chain consisting ofthe amino acid sequence of SEQ ID NO: 2 in which glutamic acid of aminoacid number 1 is modified to pyroglutamic acid and a light chainconsisting of the amino acid sequence shown by SEQ ID NO: 4;

(3) an anti-human Igβ antibody comprising a heavy chain consisting ofthe amino acid sequence of amino acid numbers 1 to 448 of SEQ ID NO: 2in which glutamic acid of amino acid number 1 is modified topyroglutamic acid and a light chain consisting of the amino acidsequence shown by SEQ ID NO: 4; and

(4) an anti-human Igβ antibody comprising a heavy chain consisting ofthe amino acid sequence shown by SEQ ID NO:2 and a light chainconsisting of the amino acid sequence shown by SEQ ID NO: 4.

In one embodiment, the pharmaceutical composition of the presentinvention comprising an anti-human Igβ antibody includes apharmaceutical composition comprising two or more anti-human Igβantibodies selected from the following (1) to (4):

(1) an anti-human Igβ antibody comprising a heavy chain consisting ofthe amino acid sequence of amino acid numbers 1 to 448 of SEQ ID NO: 6and a light chain consisting of the amino acid sequence shown by SEQ IDNO: 8;

(2) an anti-human Igβ antibody comprising a heavy chain consisting ofthe amino acid sequence of SEQ ID NO: 6 in which glutamine of amino acidnumber 1 is modified to pyroglutamic acid and a light chain consistingof the amino acid sequence shown by SEQ ID NO: 8;

(3) an anti-human Igβ antibody comprising a heavy chain consisting ofthe amino acid sequence of amino acid numbers 1 to 448 of SEQ ID NO: 6in which glutamine of amino acid number 1 is modified to pyroglutamicacid and a light chain consisting of the amino acid sequence shown bySEQ ID NO: 8; and

(4) an anti-human Igβ antibody comprising a heavy chain consisting ofthe amino acid sequence shown by SEQ ID NO:6 and a light chainconsisting of the amino acid sequence shown by SEQ ID NO: 8.

In one embodiment, the pharmaceutical composition of the presentinvention comprising an anti-human Igβ antibody includes apharmaceutical composition comprising two or more anti-human Igβantibodies selected from the following (1) to (4):

(1) an anti-human Igβ antibody comprising a heavy chain consisting ofthe amino acid sequence of amino acid numbers 1 to 448 of SEQ ID NO: 10and a light chain consisting of the amino acid sequence shown by SEQ IDNO: 12;

(2) an anti-human Igβ antibody comprising a heavy chain consisting ofthe amino acid sequence of SEQ ID NO: 10 in which glutamic acid of aminoacid number 1 is modified to pyroglutamic acid and a light chainconsisting of the amino acid sequence shown by SEQ ID NO: 12;

(3) an anti-human Igβ antibody comprising a heavy chain consisting ofthe amino acid sequence of amino acid numbers 1 to 448 of SEQ ID NO: 10in which glutamic acid of amino acid number 1 is modified topyroglutamic acid and a light chain consisting of the amino acidsequence shown by SEQ ID NO: 12; and

(4) an anti-human Igβ antibody comprising a heavy chain consisting ofthe amino acid sequence shown by SEQ ID NO: 10 and a light chainconsisting of the amino acid sequence shown by SEQ ID NO: 12.

Further, in one embodiment, the pharmaceutical composition of thepresent invention is a pharmaceutical composition described below:

a pharmaceutical composition comprising an anti-human Igβ antibodycomprising a heavy chain consisting of the amino acid sequence shown bySEQ ID NO: 6 and a light chain consisting of the amino acid sequenceshown by SEQ ID NO: 8, an anti-human Igβ antibody comprising a heavychain consisting of the amino acid sequence of amino acid numbers 1 to448 of SEQ ID NO: 6 in which glutamine of amino acid number 1 ismodified to pyroglutamic acid and a light chain consisting of the aminoacid sequence shown by SEQ ID NO: 8, and a pharmaceutically acceptableexcipient.

The addition amount of the anti-human Igβ antibody of the presentinvention in formulation varies depending on the degree of a patient'ssymptoms, the age of a patient, a dosage form of the drug to be used,the binding titer of the antibody, or the like, and for example, anaddition amount of approximately 0.001 mg/kg to 100 mg/kg can be used.

The pharmaceutical composition of the present invention can be used asan agent for treating autoimmune diseases such as systemic lupuserythematosus, rheumatoid arthritis, idiopathic thrombocytopenicpurpura, myasthenia gravis, Grave's disease, optic neuromyelitis,autoimmune hemolytic anemia, pemphigus, antiphospholipid antibodysyndrome, ANCA associated vasculitis, Sjogren's syndrome, Hashimoto'sdisease, chronic inflammatory demyelinating polyneuropathy, chronicfatigue syndrome, or the like.

The present invention includes a pharmaceutical composition forpreventing or treating systemic lupus erythematosus, rheumatoidarthritis, or idiopathic thrombocytopenic purpura comprising theanti-human Igβ antibody of the present invention. Further, the presentinvention includes a method for preventing or treating systemic lupuserythematosus, rheumatoid arthritis, or idiopathic thrombocytopenicpurpura comprising administering a therapeutically effective amount ofthe anti-human Igβ antibody of the present invention. Further, thepresent invention includes the anti-human Igβ antibody of the presentinvention for use in preventing or treating systemic lupuserythematosus, rheumatoid arthritis, or idiopathic thrombocytopenicpurpura. In addition, the present invention includes use of theanti-human Igβ antibody of the present invention for manufacture of apharmaceutical composition for preventing or treating systemic lupuserythematosus, rheumatoid arthritis, or idiopathic thrombocytopenicpurpura.

The present invention has been described and specific examples referredto for better understanding will be provided, but these are merelyexamples and the present invention is not limited thereto.

EXAMPLES

With regard to parts using commercially available kits or reagents, thetests are performed according to the attached protocol unless otherwisenoted.

Example 1 Acquisition of Human and Monkey Igβ-Flag Proteins

A protein in which a Flag tag binds to human Igβ (human Igβ-Flagprotein) and a protein in a Flag tag binds to which monkey Igβ (monkeyIgβ-Flag protein) were acquired. A human Igβ-Flag gene (SEQ ID NO: 13)was introduced into a GS vector pEE6.4 (Lonza Biologics, Inc.). A monkeyIgβ-Flag gene (SEQ ID NO: 14) was introduced into a GS vector pEE6.4(Lonza Biologics, Inc.). The respective prepared vectors weregene-transferred to FreeStyle 293 cells (Life Technologies, Inc.) usinga FreeStyle MAX Reagent (life Technologies, Inc.). Respective cells werecultured in a serum-free culture system using a FreeStyle 293 Expressionmedium (Life Technologies, Inc.) for 1 week and culture supernatantsrespectively containing human Igβ-Flag protein and monkey Igβ-Flagprotein were acquired. The proteins were purified using an anti-Flag M2antibody affinity gel (SIGMA-ALDRICH Corporation) from the acquiredculture supernatants and then used for the following test.

Example 2 Acquisition of Anti-human Igβ Antibody

In order to acquire an anti-human Igβ antibody, the human Igβ-Flagprotein and the monkey Igβ-Flag protein acquired in Example 1 wereinjected to a C3H/HeJJmsSlc-lpr/lpr mouse (Japan SLC, Inc.) togetherwith an adjuvant for causing an immune reaction to perform immunization.The mouse was immunized several times and final immunization wasperformed. According to the conventional method, a spleen and a lymphnode of the immunized mouse was extracted, and lymphocytes werecollected and cell-fused with mouse myeloma cells SP2/0 (ATCC CRL-1581),thereby preparing a hybridoma. A limiting dilution sample of thehybridoma was prepared and the hybridoma was monocloned. Respectiveclones were expanded and cultured, the culture medium was changed toHybridoma SFM (Life Technologies, Inc.), which is a serum-free culturemedium, and then the clones were cultured for 3 to 5 days. An antibodywas purified using an antibody purifying kit (Protein G Purificationkit; Proteus, Inc.) from the obtained culture supernatant.

In regard to the antibodies obtained from respective clones, the bindingactivity on human and monkey Igβ-Flag proteins and the binding activityon human and monkey B cells were evaluated. As a result, it was foundthat an antibody referred to as CL6_40 was bound to both of the humanand monkey Igβ-Flag proteins and had a high binding activity withrespect to both of the human and monkey B cells. In regard to CL6_40,genes encoding a heavy chain and a light chain from Hybridoma werecloned and sequence determination was performed.

Example 3 Preparation of Humanized Antibody

CDRs of the heavy chain and the light chain of CL6_40 were transplantedto other human antibodies, and a plurality of genes of heavy chains andlight chains of humanized antibodies were prepared. An expression vectorcomprising both genes of a heavy chain and a light chain of respectivehumanized antibodies was constructed using a GS vector (Lonza Biologics,Inc.). Specifically, genes encoding signal sequences (N. Whittle et al.,Protein Eng., Vol. 1, p. 499-505, 1987) and the constant region gene ofhuman Igγ1 (consisting of the base sequence of base numbers 358 to 1350of SEQ ID NO: 1) having amino acid mutations of S239D, H268D, and L328Wwere respectively ligated to the 5′ side and the 3′ side of the heavychain variable region genes of respective humanized antibodies, and thenthe heavy chain genes were inserted into a GS vector pEE6.4. Further,genes encoding signal sequences (N. Whittle et al., mentioned above) andthe constant region genes of a human K chain (consisting of the basesequence of base numbers 337 to 657 of SEQ ID NO: 3) were respectivelyligated to the 5′ side and the 3′ side of the light chain variableregion genes of the respective humanized antibodies, and then the lightchain genes were inserted into a GS vector pEE12.4.

The base sequence of the heavy chain of the prepared humanized antibodyCL6_40m12_DDW is shown by SEQ ID NOS: 1 and 15, the amino acid sequenceencoded by the base sequence is shown by SEQ ID NO: 2, the base sequenceof the light chain of the antibody is shown by SEQ ID NO: 3, and theamino acid sequence encoded by the base sequence is shown by SEQ ID NO:4. The heavy chain variable region shown by SEQ ID NO: 2 consists of theamino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 2, andthe CDR1, CDR2, and CDR3 of the heavy chain each consist of the aminoacid sequence of amino acid numbers 31 to 35, 50 to 65, and 98 to 108 ofSEQ ID NO: 2. The light chain variable region shown by SEQ ID NO: 4consists of the amino acid sequence of amino acid numbers 1 to 112 ofSEQ ID NO: 4, and the CDR1, CDR2, and CDR3 of the light chain eachconsist of the amino acid sequence of amino acid numbers 24 to 38, 54 to60, and 93 to 101 of SEQ ID NO: 4.

The base sequence of the heavy chain of the prepared humanized antibodyCL6_40m14_DDW is shown by SEQ ID NO: 5, the amino acid sequence encodedby the base sequence is shown by SEQ ID NO: 6, the base sequence of thelight chain of the antibody is shown by SEQ ID NO: 7, and the amino acidsequence encoded by the base sequence is shown by SEQ ID NO: 8. Thevariable region of the heavy chain shown by SEQ ID NO: 6 consists of theamino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 6, andthe CDR1, CDR2, and CDR3 of the heavy chain respectively consist of theamino acid sequence of amino acid numbers 31 to 35, 50 to 65, and 98 to108 of SEQ ID NO: 6. The variable region of the light chain shown by SEQID NO: 8 consists of the amino acid sequence of amino acid numbers 1 to112 of SEQ ID NO: 8, and the CDR1, CDR2, and CDR3 of the light chainrespectively consist of the amino acid sequence of amino acid numbers 24to 38, 54 to 60, and 93 to 101 of SEQ ID NO: 8.

The base sequence of the heavy chain of the prepared humanized antibodyCL6_40m16_DDW is shown by SEQ ID NO: 9, the amino acid sequence encodedby the base sequence is shown by SEQ ID NO: 10, the base sequence of thelight chain of the antibody is shown by SEQ ID NO: 11, and the aminoacid sequence encoded by the base sequence is shown by SEQ ID NO: 12.The variable region of the heavy chain shown by SEQ ID NO: 10 consistsof the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO:10, and the CDR1, CDR2, and CDR3 of the heavy chain respectively consistof the amino acid sequence of amino acid numbers 31 to 35, 50 to 65, and98 to 108 of SEQ ID NO: 10. The variable region of the light chain shownby SEQ ID NO: 12 consists of the amino acid sequence of amino acidnumbers 1 to 112 of SEQ ID NO: 12, and the CDR1, CDR2, and CDR3 of thelight chain respectively consist of the amino acid sequence of aminoacid numbers 24 to 38, 54 to 60, and 93 to 101 of SEQ ID NO: 12.

CDR1, CDR2, and CDR3 of each of heavy chains shown by SEQ ID NOS: 6 and10 are the same as CDR1, CDR2, and CDR3 of the heavy chain shown by SEQID NO: 2, and CDR1, CDR2, and CDR3 of each of light chains shown by SEQID NOS: 8 and 12 are the same as CDR1, CDR2, and CDR3 of the light chainshown by SEQ ID NO: 4.

In order to prepare each humanized antibody, the above-described GSvector into which the genes of the heavy chain and the light chain ofeach antibody were respectively inserted was cleaved with a restrictionenzyme by NotI and PvuI, and ligation was performed using aLigation-Convenience Kit (NIPPONGENE Co., Ltd.), thereby constructing aDouble-Gene vector into which both genes of the heavy chain and thelight chain were inserted. Next, the Double-Gene vector was transfectedusing an ExpiFectamine 293 (Life Technologies, Inc.), and cultured for 5days with respect to Expi 293 cells (Life Technologies, Inc.) culturedin an Expi 293 Expression medium (Life Technologies, Inc.) atapproximately 3000000 cells/mL. Next, purified antibodies of respectivehumanized antibodies were obtained using Protein G (GE Healthcare JapanCorporation) from the obtained culture supernatants. In regard toconstitutive expression, antibodies were expressed by transfecting theabove-described Double-Gene vector to CHO-K1SV cells (Lonza Biologics,Inc.). Then, purified antibodies of respective humanized antibodies wereobtained using MabSelect SuRe (GE Healthcare Japan Corporation) from theculture supernatants. As a result of analyzing amino acid modificationof the respective purified humanized antibodies, in most of the purifiedantibodies, deletion of lysine at the C terminal of the heavy chainoccurred in CL6_40m2_DDW, pyroglutamylation at the N terminal of theheavy chain and deletion of lysine at the C terminal of the heavy chainoccurred in CL6_40m14_DDW, and deletion of lysine at the C terminal ofthe heavy chain occurred in CL6_40m16_DDW.

Example 4 ELISA Assay with Respect to Antigen

In order to measure the antigen binding activity of the humanizedantibody, antigen ELISA was used. The human Igβ-Flag protein acquired inExample 1 was prepared with a tris-buffered saline (TBS; Wako PureChemical Industries, Ltd.) so as to have a concentration of 5000 ng/mL,added to a NUNC MaxiSorp white 384 plate (Maxisorp 384 plate: NuncCorporation) by an amount of 15 μL per well, and then solidified at roomtemperature for 1 hour. The resultant was washed with TBS-T (0.05%Tween-20 containing TBS: Wako Pure Chemical Industries, Ltd.) twice, 120μL of a blocking agent (Blocking One: Nacalai tesque, Inc.) was addedthereto, the resultant was left at room temperature for 1 hour, and thesolution was removed. A dilution series (8 steps with a finalconcentration of 0.46 ng/mL to 1 μg/mL) of respective humanizedantibodies obtained in Example 3 was prepared using a dilute solutionobtained by adding the same amount of the blocking agent and TBS andthen added thereto by an amount of 15 μL. The resultant was left at roomtemperature for 1 hour, washed with a TBS-T washing liquid three times,and 20 μL of an horseradish peroxidase (HRP)-labeled rabbit anti-humanIg antibody (Dako Ltd.) which had been diluted 3000-fold with a dilutedsolution was added thereto. Thereafter, the resultant was left at roomtemperature for 1 hour and then washed with a TBS-T washing liquid threetimes. Next, 30 μL of BM-Chemiluminescence ELISA Substrate (POD) (RocheDiagnostics Inc.) which is a chemiluminescence detection reagent wasadded thereto, and the amount of chemiluminescence thereof was measuredby an EnVision counter (PerkinElmer, Co., Ltd.). Using the same method,antigen ELISA assay was performed using the monkey Igβ-Flag proteinacquired in Example 1. When the binding activities in respectiveconcentrations of the test antibodies were calculated, the measuringamount of a well to which a test antibody was not added was set to 0%and the convergence value of the maximum activity of the test antibodywas set to 100%. The calculated binding activities were analyzed and theEC50 values of the test antibodies were calculated by fitting a curve.

As a result, the EC50 values with respect to human and monkey Igβ-Flagproteins of CL6_40m12_DDW were respectively 128 ng/mL and 183 ng/mL. TheEC50 values with respect to human and monkey Igβ-Flag proteins ofCL6_40m14_DDW were respectively 100 ng/mL and 106 ng/mL. The EC50 valueswith respect to human and monkey Igβ-Flag proteins of CL6_40m16_DDW wererespectively 132 ng/mL and 118 ng/mL. It was confirmed that all of therespective humanized antibodies had high binding activities with respectto both of the human and monkey Igβ-Flag proteins.

Example 5 FACS Analysis with Respect to Human and Monkey PBMC

In order to evaluate the binding activities of humanized antibodies withrespect to human and monkey cells, Fluorescence Activated Cell Sorting(FACS) analysis was performed on human and monkey PBMC with an index ofCD20 which is a B cell marker using B cells contained in the PBMC as atarget. The monkey PBMC was prepared by diluting the blood of a monkeyin the same amount of PBS (Life Technologies, Inc.), laminating thediluted blood on the same amount of Ficoll (GE Healthcare JapanCorporation), and performing a centrifugal treatment at room temperatureand at 1500 rpm for 30 minutes. Next, human PBMC (AllCells, Inc.) ormonkey PBMC was seeded by an amount of 200000 per well in a 96-wellplate (Greiner Bio-One) in a state of being suspended in 30 μL of StainBuffer (Becton, Dickinson Company). A dilution series (4 steps with afinal concentration of 0.03 ng/mL to 30 μg/mL) of each of the humanizedantibodies acquired in Example 3 was prepared using Stain Buffer and 30μL of the dilution series was added thereto. The resultant was left onice for 30 minutes, washed with Stain Buffer three times, and 40 μL of asolution having a phycoerythrin-labeled goat anti-human IgG Fcγ fragment(JACKSON, Inc.) which was diluted 200-fold with Stain Buffer and anallophycocyanin-labeled mouse anti-CD20 antibody (Becton, DickinsonCompany) diluted 8-fold with Stain Buffer was added thereto. Theresultant was left on ice for 30 minutes and washed with Stain Buffertwice, the fluorescence intensity was measured using FACSArray (Becton,Dickinson Company), and then the mean fluorescence intensity: MFI) wascalculated. FlowJo (TOMY DIGITAL BIOLOGY Co., Ltd.) was used foranalysis.

As a result, it was confirmed that all of the respective humanizedantibodies had high binding activities with respect to both of the humanand monkey B cells.

Example 6 Evaluation of Anti-IgM Antibody-induced Cell ProliferationActivity

In order to evaluate the inhibitory effect of a humanized antibodieswith respect to activation of human B cells due to BCR stimulation,anti-IgM antibody-induced cell proliferation activity in human B cellswas evaluated. The anti-IgM antibody activates B cells by allowing BCRto aggregate. An antibody binding to both of BCR and FcγRIIb mobilizesFcγRIIb to BCR and thus proliferation of B cells can be inhibited. Inthis Example, anti-CD19 S267E/L328F (Patent Document 2) was used as acomparative antibody. As a control antibody, a human IgG1 antibody(anti-KLH Ab) against KLH (keyhole limpet hemocyanin) which is anantigen not existing in a living body was used (WO 2013/094723). Next,human B cells (AllCells, Inc.) were seeded by an amount of 30000 perwell in a 96-well plate (Iwaki, Co., Ltd.) using a 60 μL of RPMI culturemedium (SIGMA-ALDRICH Corporation). Subsequently, dilution series (3steps with a final concentration of 0.3 ng/mL to 30 μg/mL) of therespective full human antibodies acquired in Example 3, anti-CD19S267E/L328F, or anti-KLH Ab were prepared and added thereto by an amountof 20 μL using the RPMI culture medium. 20 μL of the anti-IgM antibody(JACKSON, Inc.) prepared such that the final concentration thereof inthe RPMI culture medium was adjusted to 5 μg/mL was added and incubatedin a CO₂ incubator for 4 days. Next, cell proliferation analysis wasperformed using CellTiter-Glo (Promega K.K.). In addition, in Examplehere, a test antibody non-added/anti-IgM antibody non-added group and atest antibody non-added/anti-IgM antibody added group were respectivelyprepared as a negative control and a positive control and then a testwas performed. Respective test antibodies were tested in duplicate.

FIG. 1 shows the results of the proliferation rate of human B cells. Theproliferation rates of a test antibody administration group wascalculated by setting a test antibody non-added/anti-IgM antibodynon-added group as a negative control (proliferation rate: 0%) and atest antibody non-added/anti-IgM antibody added group as a positivecontrol (proliferation rate: 100%). This means that the inhibitoryactivity with respect to BCR of a test antibody is stronger when thevalue of the proliferation rate thereof is smaller.

As shown in FIG. 1, while the proliferation rate in 30 μg/mL ofanti-CD19 S267E/L328F was 52.3%, the proliferation rates in 30 μg/mL ofCL6_40m12_DDW, CL6_40m14_DDW, and CL6_40m16_DDW were respectively 2.8%,20.2%, and 23.2%. Therefore, it is evident that all of theabove-described full human antibodies have strong inhibitory activitieswith respect to anti-IgM antibody-induced cell proliferation in human Bcells compared to anti-CD19 S267E/L328F.

Example 7 Evaluation of Drug Efficacy in Human PBMC Transfer NOG MouseModel

For the purpose of verifying the effectiveness of a humanized antibodywith respect to in vivo antibody production, an action of variousantibodies in administration for treatment with respect to an increasein human antibody titers induced by transferring human PBMC into an NOGmouse was evaluated. In the present model, it is considered that thehuman B cells violently activated by foreign object (mouse) recognitiondifferentiate into plasma (blast) cells in the body of the mouse, andthe present model is appropriate for evaluating a pharmacological actionof a test drug with respect to the activity of human B series cells.

Human PBMC (AllCells, Inc.) was suspended at 10000000 cells/mL in PBS(Wako Pure Chemical Industries, Ltd.) and administered to the tail veinof a 11-week-old male NOG mouse (In-vivo Science, Inc.) by an amount of0.25 mL (2500000 cells). On the 34^(th) day (34^(th) day after PBMCtransfer), the weight was measured and blood was sampled. The plasmahuman IgM and IgE antibody titer was measured using ELISA (BethylLaboratories, Inc.). Grouping was performed based on the plasma humanIgM, the IgE antibody titer, and the weight data.

In this Example, as a comparative antibody, anti-CD19 S267E/L328F wasused. As a control antibody, anti-KLH Ab was used. 10 mg/10 mL/kg of atest antibody was administered to a mouse by subcutaneous administrationon the 35^(th) and the 42^(nd) days. Blood sampling was performed on the42^(nd) and the 49^(th) days and the plasma human IgM and IgE antibodytiter was measured using ELISA (Bethyl Laboratories, Inc.). The test wasperformed in a unit of a group of 4 or 5 animals. The test results areshown by “average value±standard error.” A significant difference testof an anti-KLH Ab group and various test antibody groups was performedusing a Student's t-test, and a case where the p value was less than0.05 was regarded as statistically significant. The above-described testwas performed using a GraphPad Prism (version 5.04).

FIG. 2 shows an action of a test antibody with respect to the plasmahuman IgM antibody titer. The plasma human IgM antibody titer wassignificantly decreased by CL6_40m12_DDW and CL6_40m14_DDW compared toanti-KLH Ab. An action of decreasing CL6_40m12_DDW and CL6_40m14_DDWwith respect to the plasma human IgM antibody titer was expressedexceedingly early and recognized from the first week after theadministration was started (42^(nd) day). Meanwhile, in anti-CD19S267E/L328F, an action of a decrease with respect to the plasma humanIgM antibody titer was significant only after 2 weeks afteradministration was started (49^(th) day).

Next, FIG. 3 shows an action of a test antibody with respect to theplasma human IgE antibody titer. In CL6_40m12_DDW and CL6_40m14_DDW, theplasma human IgE antibody titer was rapidly and significantly decreasedcompared to anti-KLH Ab. Meanwhile, in anti-CD19 S267E/L328F, the plasmahuman IgE antibody titer was not decreased.

As shown in FIGS. 2 and 3, it is evident that both of theabove-described CL6_40m12_DDW and CL6_40m14_DDW have strong inhibitoryactivities with respect to an increase in the human antibody titercompared to anti-CD19 S267E/L328F.

Example 8 Evaluation of Drug Efficacy in Monkey TTx AntigenSensitization Model

TTx antigen-specific IgG was produced by sensitizing an adsorbed tetanustoxoid (TTx) antigen to a monkey once. In the present model, the totalantibody titers in plasma can be evaluated in addition to TTxantigen-specific IgG in plasma. Accordingly, in the present model,safety can be evaluated in addition to effectiveness thereof whenautoimmune diseases are treated.

Using a male cynomolgus monkey (producing area: China, 3 years old orolder), 2 mg/kg to 5 mg/kg (0.05 ml/kg: USP Corporation) of zolazepamhydrochloride and 2.5 mg/kg to 5 mg/kg of tiletamine hydrochloride weremixed under anesthesia and TTx was sensitized (the sensitization day wasset to Day 0). The sensitization of TTx was performed by injecting 0.6mL/monkey of tetanus toxoid (TTx, 10 Lf/mL, Denka Seika Co., Ltd.) tothigh muscle and 0.6 mL/monkey (respectively 50 μL to 12 places) to theintradermal back portion. As treated groups, a Vehicle group (solvent(20 mM of sodium citrate buffer/120 mM, NaCl (pH: 6.0); KOHJIN BIO Co.,Ltd.) 1 mL/kg, n=3) and an antibody administration group (10 mg/1 mL/kg,humanized antibody CL6_40m14_DDW (diluted with solvent), n=3) were used.The timing of administration was set to the 14^(th) day after TTxsensitization and the administration to the vein was performed with adosage of 1 mL/kg when awakening.

After CL6_40m14_DDW was administered to the above-described cynomolgusmonkey, blood was sampled with time, for example, after 4 hours, 72hours, 168 hours, and 336 hours, and was subjected to a centrifugaltreatment, and then plasma was recovered. The concentration of drugs inthe plasma was measured using Gyrolab™xP workstation (Gyros AB). As themethod and the disc, 200-3W-001-A and Bioaffy 200 compact discs (GyrosAB) were used. In addition, as a solidified antigen and a detectionantibody, biotin-labeled Recombinant Human CD79B (Novoprotein, Inc.) andalexa-labeled Goat Anti-Human IgG (Southern Biotechnology Associates,Inc.) were used. As listed in Table 1, the concentration of drugs inplasma of CL6_40m14_DDW was maintained during the evaluation period ofthe model.

Table 1: Transition of concentration of drugs in plasma with respect tohumanized anti-Igβ antibody in monkey

TABLE 1 Concentration of Concentration of Concentration of drugs inplasma drugs in plasma drugs in plasma of individual 1 of individual 2of individual 3 (μg/mL) (μg/mL) (μg/mL) After 4 252.111 263.242 258.271hours After 72 143.586 144.095 114.605 hours After 168 120.070 112.86595.848 hours After 336 76.854 62.281 53.305 hours

Blood was sampled from the above-described cynomolgus monkey with timeon the 13^(th) day (13 days after the cynomolgus was immunized byadsorbed tetanus toxoid), the 14^(th) day, the 17^(th) day, the 21^(st)day, and the 28^(st) day, a centrifugal treatment was carried out, andplasma was recovered. In order to measure anti-adsorbed tetanus toxoid(anti-TTx IgG) in the recovered plasma, the antigen ELISA was used. Theadsorbed tetanus toxoid (Denka Seika Co., Ltd.) was diluted 20-fold witha phosphate-buffered saline (PBS; Wako Pure Chemical Industries, Ltd.),was added to a NUNC MaxiSorp 96 plate (Maxisorp 96 plate: NuncCorporation) by an amount of 100 μL per well, and then solidified at 4°C. for one night. The resultant was washed with PBS-T (0.05% Tween-20containing PBS: Thermo Scientific, Inc.) four times, 200 μL of ablocking agent (Blocker Casein In PBS; Life Technologies, Inc.) wasadded thereto, the resultant was left at room temperature for 2 hour,and the solution was removed. Next, 100 μL of the recovered plasma and100 μL of a sample for a calibration curve were respectively addedthereto. As the sample for a calibration curve, a sample mixed withplasma collected 21 days and 23 days later from immunization of thecynomolgus monkey by adsorbed tetanus toxoid was used, the amountthereof was adjusted to 100 U/mL, and a dilution series (0.488 mU/mL to500 mU/mL) prepared using a blocking agent as a diluted solution wasused. The resultant was left at room temperature for 2 hour, washed witha PBS-T washing liquid four times, and 100 μL of a horseradishperoxidase (HRP)-labeled goat anti-monkey IgG antibody (Nordic, Inc.)which had been diluted 3000-fold with a blocking agent was addedthereto. Thereafter, the resultant was left at room temperature for 1hour and then washed with the PBS-T washing liquid four times. Next,measurement was performed using TMB Microwell Peroxidase SubstrateSystem (KPL, Inc.). The absorbance thereof was measured by SpectraMax(Molecular Devices, Inc.).

FIG. 4 shows an action of the test antibody with respect to the adsorbedtetanus toxoid antibody titer in plasma. The adsorbed tetanus toxoidantibody titer in plasma (anti-TTX IgG) was decreased in CL6-40m14_DDWcompared to the Vehicle group.

The total antibody titers (IgM, IgA, and IgG) in plasma recovered fromthe cynomolgus monkey on the 14^(th) day, the 17^(th) day, the 21^(st)day, and the 28 day were measured using the following method. A rabbitanti-monkey IgM polyclonal antibody (COVANCE, Inc.) and a rabbitanti-human IgA polyclonal antibody (Bethyl Laboratories, Inc.) werediluted 100-fold, 500-fold, and 1000-fold with a phosphate-bufferedsaline (PBS: Wako Pure Chemical Industries, Ltd.), added to a NUNCMaxiSorp 96 plate (Maxisorp 96 plate: Nunc Corporation) by an amount of100 μL, and then solidified at 4° C. for one night. The resultant waswashed with PBS-T (0.05% Tween-20 containing PBS: Thermo Scientific,Inc.) four times, 200 μL of a blocking agent (Blocker Casein In PBS;Life Technologies, Inc.) was added thereto, and the resultant was leftat room temperature for 1 hour and washed with the PBS-T washing liquidfor times. A dilution series of a sample for a calibration curve andcollected plasma of a monkey was prepared using a blocking agent as adiluted solution and 100 μL of the dilution series was added thereto. Asthe sample for a calibration curve, plasma prepared from a normalcynomolgus monkey was diluted and then used. The resultant was left atroom temperature for 2 hour, washed with a PBS-T washing liquid fourtimes, and an horseradish peroxidase (HRP)-labeled anti-monkey IgMantibody (KPL, Inc.), an horseradish peroxidase (HRP)-labeled anti-humanIgA antibody (Bethyl Laboratories, Inc.), and a horseradish peroxidase(HRP)-labeled anti-monkey IgG antibody (KPL, Inc.) were respectivelydiluted 1000-fold, 5000-fold, and 3000-fold with a blocking agent andadded thereto by an amount of 100 μL respectively. Thereafter, theresultant was left at room temperature for 2 hours and then washed witha PBS-T washing liquid four times. Next, measurement was performed usingTMB Microwell Peroxidase Substrate System (KPL, Inc.). The absorbancethereof was measured by SpectraMax (Molecular Devices, Inc.).

FIGS. 5, 6, and 7 show actions of the test antibodies with respect tothe total antibody titers (IgM, IgA, and IgG) in plasma. CL6_40m14_DDWdid not affect the total antibody titers (IgM, IgA, and IgG) in plasmacompared to the Vehicle group.

From the results described above, it is evident that CL6_40m14_DDWsuppresses an antigen-specific antibody without affecting the totalantibody titers in plasma. Further, it is also evident thatCL6_40m14_DDW has an excellent profile in terms of safety in addition toeffectiveness at the time of treatment of autoimmune diseases.

INDUSTRIAL APPLICABILITY

The anti-human Igβ antibody of the present invention is useful forpreventing and treating autoimmune diseases. Further, thepolynucleotide, the expression vectors, the transformed host cell, andthe methods for producing the antibody of the present invention areuseful for producing the anti-human Igβ antibody.

Sequence List Free Text

In the number heading <223> of the sequence list, description of“Artificial Sequence” is made. Specifically, the base sequences shown bySEQ ID NOS: 1 and 3 of the sequence list are the base sequences of theheavy chain and the light chain of the CL6_40m12_DDW, respectively, andthe amino acid sequences shown by SEQ ID NOS: 2 and 4 are the amino acidsequences of the heavy chain and the light chain encoded by the SEQ IDNOS: 1 and 3, respectively. The base sequences shown by SEQ ID NOS: 5and 7 of the sequence list are the base sequences of the heavy chain andthe light chain of the CL6_40m14_DDW, respectively, and the amino acidsequences shown by SEQ ID NOS: 6 and 8 are the amino acid sequences ofthe heavy chain and the light chain encoded by the SEQ ID NOS: 5 and 7,respectively. The base sequences shown by SEQ ID NOS: 9 and 11 of thesequence list are the base sequences of the heavy chain and the lightchain of the CL6_40m16_DDW, respectively, and the amino acid sequencesshown by SEQ ID NOS: 10 and 12 of the sequence list are the amino acidsequences of the heavy chain and the light chain encoded by the SEQ IDNOS: 9 and 11, respectively. The base sequence shown by SEQ ID NO: 13 ofthe sequence list is the base sequence of the human Igβ-Flag gene andthe base sequence shown by SEQ ID NO: 14 of the sequence list is thebase sequence of the monkey Igβ-Flag gene. The base sequence shown bySEQ ID NO: 15 of the sequence list is the base sequence of the heavychain of the CL6_40m12_DDW.

The invention claimed is:
 1. An anti-human Igβ antibody comprising a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence of amino acid numbers 50 to 65 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence of amino acid numbers 98 to 108 of SEQ ID NO: 2, a light chain variable region comprising CDR1 consisting of the amino acid sequence of amino acid numbers 24 to 38 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of amino acid numbers 54 to 60 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence of amino acid numbers 93 to 101 of SEQ ID NO: 4, and a heavy chain constant region which is a human Igγl constant region having amino acid mutations of S239D, H268D, and L328W.
 2. The anti-human Igβ antibody according to claim 1, selected from the group consisting of the following (a) to (d): (a) an anti-human Igβ antibody comprising a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 6, a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 112 of SEQ ID NO: 8, and a heavy chain constant region which is a human Igγl constant region having amino acid mutations of S239D, H268D, and L328W; (b) an anti-human Igβ antibody comprising a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 2, a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 112 of SEQ ID NO: 4, and a heavy chain constant region which is a human Igγl constant region having amino acid mutations of S239D, H268D, and L328W; (c) an anti-human Igβ antibody comprising a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 119 of SEQ ID NO: 10, and a light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 112 of SEQ ID NO: 12, and a heavy chain constant region which is a human Igγl constant region having amino acid mutations of S239D, H268D, and L328W; and (d) an anti-human Igβ antibody which is derived from posttranslational modification of the anti-human Igβ antibody of any one of (a) to (c) above, wherein the posttranslational modification is pyroglutamylation at the N terminal of the heavy chain variable region and/or deletion of lysine at the C terminal of the heavy chain.
 3. The anti-human Igβ antibody according to claim 2, selected from the group consisting of the following (a) to (d): (a) an anti-human Igβ antibody comprising a heavy chain consisting of the amino acid sequence shown by SEQ ID NO: 6 and a light chain consisting of the amino acid sequence shown by SEQ ID NO: 8; (b) an anti-human Igβ antibody comprising a heavy chain consisting of the amino acid sequence shown by SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown by SEQ ID NO: 4; (c) an anti-human Igβ antibody comprising a heavy chain consisting of the amino acid sequence shown by SEQ ID NO: 10 and a light chain consisting of the amino acid sequence shown by SEQ ID NO: 12; and (d) an anti-human Igβ antibody which is derived from posttranslational modification of the anti-human Igβ antibody of any one of (a) to (c) above, wherein the posttranslational modification is pyroglutamylation at the N terminal of the heavy chain variable region and/or deletion of lysine at the C terminal of the heavy chain.
 4. The anti-human Igβ antibody according to claim 2, selected from the group consisting of the following (a) to (f): (a) an anti-human Igβ antibody comprising a heavy chain consisting of the amino acid sequence shown by SEQ ID NO: 6 and a light chain consisting of the amino acid sequence shown by SEQ ID NO: 8; (b) an anti-human Igβ antibody comprising a heavy chain consisting of the amino acid sequence of amino acid numbers of 1 to 448 of SEQ ID NO: 6 in which glutamine of amino acid number 1 is modified to pyroglutamic acid and a light chain consisting of the amino acid sequence shown by SEQ ID NO: 8; (c) an anti-human Igβ antibody comprising a heavy chain consisting of the amino acid sequence shown by SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown by SEQ ID NO: 4; (d) an anti-human Igβ antibody comprising a heavy chain consisting of the amino acid sequence of amino acid numbers 1 to 448 of SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown by SEQ ID NO: 4; (e) an anti-human Igβ antibody comprising a heavy chain consisting of the amino acid sequence shown by SEQ ID NO: 10 and a light chain consisting of the amino acid sequence shown by SEQ ID NO: 12; and (f) an anti-human Igβ antibody comprising a heavy chain consisting of the amino acid sequence of amino acid numbers 1 to 448 of SEQ ID NO: 10 and a light chain consisting of the amino acid sequence shown by SEQ ID NO:
 12. 5. The anti-human Igβ antibody according to claim 4, wherein the anti-human Igβ antibody comprises a heavy chain consisting of the amino acid sequence shown by SEQ ID NO: 6 and a light chain consisting of the amino acid sequence shown by SEQ ID NO:
 8. 6. The anti-human Igβ antibody according to claim 4, wherein the anti-human Igβ antibody comprises a heavy chain consisting of the amino acid sequence of amino acid numbers of 1 to 448 of SEQ ID NO: 6 in which glutamine of amino acid number 1 is modified to pyroglutamic acid and a light chain consisting of the amino acid sequence shown by SEQ ID NO:
 8. 7. The anti-human Igβ antibody according to claim 2, wherein the antibody is fused to another peptide.
 8. The anti-human Igβ antibody according to claim 2, wherein the antibody is modified with polyethylene glycol, sugar chains, phospholipids, liposomes, or low-molecular compounds.
 9. A pharmaceutical composition comprising: an anti-human Igβ antibody according to claim 1, and a pharmaceutically acceptable excipient.
 10. A pharmaceutical composition comprising an anti-human Igβ antibody selected from the group consisting of the following (a) to (c), and a pharmaceutically acceptable excipient: (a) an anti-human Igβ antibody comprising a heavy chain consisting of the amino acid sequence shown by SEQ ID NO: 6 and a light chain consisting of the amino acid sequence shown by SEQ ID NO: 8, and/or an anti-human Igβ antibody comprising a heavy chain consisting of the amino acid sequence of amino acid numbers of 1 to 448 of SEQ ID NO: 6 in which glutamine of amino acid number 1 is modified to pyroglutamic acid and a light chain consisting of the amino acid sequence shown by SEQ ID NO: 8; (b) an anti-human Igβ antibody comprising a heavy chain consisting of the amino acid sequence shown by SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown by SEQ ID NO: 4, and/or an anti-human Igβ antibody comprising a heavy chain consisting of the amino acid sequence of amino acid numbers 1 to 448 of SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown by SEQ ID NO: 4; and (c) an anti-human Igβ antibody comprising a heavy chain consisting of the amino acid sequence shown by SEQ ID NO: 10 and a light chain consisting of the amino acid sequence shown by SEQ ID NO: 12, and/or an anti-human Igβ antibody comprising a heavy chain consisting of the amino acid sequence of amino acid numbers 1 to 448 of SEQ ID NO: 10 and a light chain consisting of the amino acid sequence shown by SEQ ID NO:
 12. 